Orosomucoid has a cAMP-dependent effect on human endothelial cells and inhibits the action of histamine.

The plasma protein orosomucoid (alpha(1)-acid glycoprotein) has previously been shown to constitute a critical component of the capillary barrier. The protein has also been suggested to act as an anti-inflammatory mediator in a diversity of experimental situations. Recently we reported that orosomucoid is synthesized by the microvascular endothelial cells per se. In the present study, the effects of orosomucoid on primary cultures of human umbilical vein endothelial cells (HUVEC) were studied using the Cytosensor microphysiometer. We found that 1) orosomucoid (0.01 g/l) increased the metabolic activity of HUVEC as reflected by the increased acidification rate of +14 +/- 1%; 2) pretreatment with 0.5 mM 8-bromo-cAMP for 20 min markedly and reversibly inhibited the effect of orosomucoid, whereas 8-bromo-cGMP did not; 3) histamine elicited a dose-dependent response that was abolished by pretreatment with either cAMP or cGMP; and finally, 4) pretreatment of HUVEC for 6 min with orosomucoid (0.01 g/l) inhibited the action of histamine. In summary, this is the first report demonstrating that orosomucoid affects human endothelial cells and that it does so by using cAMP as a second messenger. This provides an explanation for previous findings of anti-inflammatory effects of the protein and shows that orosomucoid affects the endothelium during both normal and pathophysiological conditions.

Vertebral plasma cell tumors in 8 dogs.

The case histories of 8 dogs with spinal pain and neurologic deficits associated with vertebral plasma cell tumor are reviewed. Four dogs had solitary plasmacytoma, 3 had multiple myeloma, and 1 dog had 2 vertebral lesions with no evidence of disseminated disease. Four dogs were treated: 2 with multiple myeloma received chemotherapy only and survived 17 and 26 months, respectively. Two dogs with solitary plasmacytomas of the spine had chemotherapy and radiotherapy: the 1st survived 4 months and was euthanized after developing radiation myelopathy; the 2nd survived 65 months before developing multiple myeloma. The diagnosis of solitary plasmacytoma of the spine versus multiple myeloma is discussed.

Polyostotic lymphoma in a young dog: a case report and literature review.

An eight-month-old female German shepherd dog had pathological fractures affecting the distal radius and ulna and ribs. Radiographically, there were bilaterally symmetrical osteolytic lesions affecting the metaphyses of multiple long bones, ribs and skull and the dog had splenomegaly. Histologically, the spleen, thymus and bones were infiltrated with large lymphoblastic cells with a high mitotic rate; the diagnosis was lymphoma. Lymphoma primarily affecting bone is an uncommon diagnosis in the dog but it should be considered in young animals with osteolytic lesions affecting multiple bones.

Serotype-specific immunoglobulin G antibody responses to pneumococcal polysaccharide vaccine in children with sickle cell anemia: effects of continued penicillin prophylaxis.

OBJECTIVES: (1) To determine serotype-specific IgG antibody responses to reimmunization with pneumococcal polysaccharide vaccine at age 5 years in children with sickle cell anemia and (2) to determine whether continued penicillin prophylaxis had any adverse effects on these responses. STUDY DESIGN: Children with sickle cell anemia, who had been treated with prophylactic penicillin for at least 2 years before their fifth birthday, were randomly selected at age 5 years to continue penicillin prophylaxis or to receive placebo treatment. These children had been immunized once or twice in early childhood with pneumococcal polysaccharide vaccine and were reimmunized at the time of randomization. RESULTS: Serotype-specific IgG antibody responses to reimmunization varied according to pneumococcal serotype but in general were mediocre or poor; the poorest response was to serotype 6B. The antibody responses were similar in subjects with continued penicillin prophylaxis or placebo treatment, and in subjects who received one or two pneumococcal vaccinations before reimmunization. The occurrence of pneumococcal bacteremia was associated with low IgG antibody concentrations to the infecting serotype. CONCLUSIONS: Reimmunization of children with sickle cell anemia who received pneumococcal polysaccharide vaccine at age 5 years induces limited production of serotype-specific IgG antibodies, regardless of previous pneumococcal vaccine history. Continued penicillin prophylaxis does not interfere with serotype-specific IgG antibody responses to reimmunization.

The pentameric structure of IgM is necessary to enhance opsonization of Bacteroides thetaiotaomicron and Bacteroides fragilis via the alternative complement pathway.

Studies were conducted to investigate the mechanisms by which natural IgM antibodies act together with the alternative complement pathway to promote opsonization and adherence of encapsulated Bacteroides thetaiotaomicron and Bacteroides fragilis to polymorphonuclear leukocytes (PMN). A model system consisting of the six isolated proteins of the alternative pathway was used. A comparison of the opsonic effects of pentameric and monomeric forms of isolated normal IgM demonstrated that, although the monomeric form bound to Bacteroides as effectively as the pentameric form and promoted complement deposition to the same extent, it was unable to enhance alternative pathway-dependent opsonization and adherence of Bacteroides to PMN. When opsonization was performed in two steps with pentameric IgM added either before or after alternative pathway components, a marked enhancement of adherence to PMN was observed only in the former case, suggesting IgM must act prior to complement to be effective. Electron microscopic studies demonstrated that, when added with complement, pentameric IgM, but not monomeric IgM, stabilized the bacterial capsule to the dehydration in dimethylformamide used for embedding in Lowicryl K4M. A strong correlation was observed between capsular stability and ability to be bound by PMN. The results suggest that pentameric IgM alters the structure of capsular components, perhaps through crosslinking, and this is in turn facilitates interaction of C3bi and C3b with CR3 and CR1, their respective receptors on PMN.

The alternative complement pathway promotes IgM antibody-dependent and -independent adherence of Bacteroides to polymorphonuclear leukocytes through CR3 and CR1.

Natural immunoglobulin M (IgM) antibodies act synergistically with the alternative complement pathway to promote opsonization and adherence of Bacteroides thetaiotaomicron and Bacteroides fragilis to polymorphonuclear leukocytes (PMNs). This study characterizes the PMN receptors involved in adherence of Bacteroides opsonized by the alternative pathway and determines the effect of natural IgM antibodies on receptor involvement and on the molecular form of C3 deposited on the bacteria. A model system consisting of the six isolated proteins of the alternative pathway with or without supplemental isolated normal IgM was used for opsonization, and results were compared to those obtained with serum. The results demonstrate that the alternative pathway promotes adherence of Bacteroides to PMNs through complement receptors 1 and 3 (CR3), and epitopes in CR3 besides those mediating iC3b binding do not participate. The results also demonstrate that natural IgM antibodies do not influence the character of the ligands or receptors involved in this interaction.

Down-regulation of chemotaxis of polymorphonuclear leukocytes following thermal injury involves two distinct mechanisms.

Thermal injury induces a depression of major effector functions of polymorphonuclear leukocytes (PMNL) that contributes to the increased susceptibility to bacterial infection associated with severe injury. In a study on chemotactic alterations in PMNL induced by thermal injury in a well-characterized guinea pig model, a concomitant reduction in the chemotactic response of PMNL to zymosan-activated serum (ZAS) and FMLP was seen early after thermal injury in temporal association with the previously reported bactericidal defect and depression of superoxide anion production. Unlike the bactericidal defect, the chemotactic alterations were not directly linked to the marked elevation of intracellular cAMP in PMNL associated with thermal injury. Two mechanisms (adaptation and desensitization) were shown to be involved in the reduction of chemotactic responses of PMNL to FMLP and ZAS, respectively. Adaptation appears to be a protective response of PMNL to thermal injury unassociated with receptor down-regulation.

Circulating factors contribute to elevation of intracellular cyclic-3',5'-adenosine monophosphate and depression of superoxide anion production in polymorphonuclear leukocytes following thermal injury.

We have previously demonstrated that bactericidal activity and superoxide anion (O2-) production are depressed concomitantly in polymorphonuclear leukocytes (PMNs) following thermal injury in a guinea pig model, and the bactericidal defect is related to elevation of intracellular cyclic-3',5'-adenosine monophosphate (cAMP). The purpose of the present investigation was to determine the relationship between elevation of intracellular cAMP and depression of O2- production in PMNs following thermal injury and determine the involvement of circulating factors in the development of these alterations. The kinetics of O2- production and dose responses to formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA) were depressed in peripheral PMNs following thermal injury in this experimental model. Sera obtained during the period of PMN dysfunction induced depression of O2- production in response to fMLP and elevation of intracellular cAMP in normal PMNs. Pretreatment of normal PMNs with nonsteroidal anti-inflammatory drugs (NSAID; indomethacin or piroxicam) inhibited the elevation of intracellular cAMP mediated by sera from the injured animals but had no effect on the depression of O2- production observed under similar conditions. Treatment of PMNs from injured animals with NSAID under conditions known to reduce the cAMP content of the cells and correct the bactericidal defect did not normalize O2- production. Studies utilizing sera from two thermally injured patients confirmed findings in the guinea pig model of serum-mediated elevation of intracellular cAMP and depression of O2- production in normal PMNs and effects observed with NSAID. These results suggest that circulating factors contribute to the elevation of intracellular cAMP and depression of O2- production in PMNs following thermal injury. Whereas the increase in intracellular cAMP may be involved in the depression of O2- production, our results suggest that there is not a direct link between these alterations.

The effect of immunotherapy on eosinophil accumulation and production of eosinophil chemotactic activity in the lung of subjects with asthma during natural pollen exposure.

Two groups of birch pollen--allergic patients with seasonal rhinoconjunctivitis and asthma were followed during two consecutive birch-pollen seasons, one group, N = 10, during a season with high pollen load, and one group, N = 15, during a season of low pollen load. Half the patients were treated with immunotherapy (IT) for 3 and 4 years, respectively. The other half of the patients served as control group (non-IT). Bronchoalveolar lavage (BAL) was performed once before each season and once during the pollen season. Eosinophil (EOS) numbers in BAL were increased (p less than 0.01) during the season with high pollen load but not in the season with a low pollen load, and this increment was absent in the IT-treated group. Also, the EOS cationic protein levels were raised in the non-IT-treated group during the season with a high pollen load. The levels of EOS and neutrophil chemotactic activity were raised in BAL in both seasons in the non-IT-treated group compared with the IT-treated group (p less than 0.02, p less than 0.003, p less than 0.04, and p less than 0.005 in high- and low-load pollen season, respectively). Serum and BAL eosinophil chemotactic activity (ECA) were positively correlated (p less than 0.001). We conclude that there is an influx of active EOSs into the lung of pollen-allergic patients with asthma during a pollen season, which may be abrogated by IT. Furthermore, the generation of ECA appears to be an extremely sensitive marker of antigenic exposure, and the potent inhibition of the generation of ECA by IT may provide a clue as to the mechanism of this treatment.

Complement is activated in the upper respiratory tract during influenza virus infection.

The purpose of this pilot study was to determine whether complement is activated in the upper respiratory tract during experimental influenza virus infection in human volunteers. Seven subjects were challenged with influenza A/Bethesda/1/85 (H3N2), and four subjects received placebo. C3a and C5a concentrations were measured by radioimmunoassay in nasal lavage fluids before challenge and for 8 days after challenge. A significant increase (p less than 0.05) in C3a and C5a concentrations was demonstrated in lavage fluids from subjects who developed influenza illness as compared with uninfected control subjects and infected subjects who remained asymptomatic. Maximal levels of C3a and C5a were detected during the recovery phase of illness. These results suggest that complement is activated in the airway in response to influenza illness.

Effects of an inhaled corticosteroid, budesonide, on alveolar macrophage function in smokers.

Selected functions of alveolar macrophages obtained by bronchoalveolar lavage of 12 healthy smokers were examined before and after eight weeks' treatment with an inhaled glucocorticosteroid, budesonide (400 micrograms twice daily). After budesonide treatment spontaneous as well as opsonised zymosan triggered prostaglandin E2 (PGE2) secretion from harvested cells was reduced; no such reduction in opsonised zymosan triggered leukotriene B4 (LTB4) production was observed. Neither the capacity to phagocytose opsonised yeast particles nor the superoxide radical generation triggered by the calcium ionophore A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA), or opsonised zymosan ex vivo were more than marginally affected by the glucocorticosteroid treatment in vivo. Lavage fluid concentrations of angiotensin converting enzyme (ACE), however, after treatment were twice those before treatment and concentrations of fibronectin were reduced to half. Albumin concentrations in lavage fluid were not affected by the glucocorticosteroid treatment. In separate experiments treatment of alveolar macrophages with 10(-7) or 10(-6) M budesonide overnight in vitro did not affect their superoxide radical or PGE2 generation but significantly blocked LTB4 release. These data indicate that inhaled gluco-corticosteroid treatment may affect synthesis or release (or both) of ACE and fibronectin by alveolar macrophages from healthy smokers whereas other functions of these cells, such as the generation of reactive oxygen derived products ex vivo, are only marginally affected.

Role of humoral factors in host resistance to the Bacteroides fragilis group.

The alternative complement pathway and antibody play an important role in host resistance to members of the Bacteroides fragilis group. Clinical isolates of the B. fragilis group are typically resistant to the bactericidal action of serum. Naturally occurring antibodies in serum directed against these bacteria belong primarily to the IgM class. These antibodies are not required for activation of the alternative pathway by the bacteria but rather potentiate alternative pathway activation. Activation of the alternative pathway by the bacteria leads to the generation of chemotactic activity for neutrophils and to C3 deposition on the bacterial surfaces. Both iC3b, the predominant molecular form of the bacteria-bound C3, and IgM antibody are necessary for phagocytosis and killing of the bacteria by neutrophils. IgM acts synergistically with iC3b to facilitate adherence of the bacteria to the neutrophils. Certain Bacteroides strains require additional as yet uncharacterized auxiliary opsonins for maximal adherence to neutrophils.

Bactericidal defect of neutrophils in a guinea pig model of thermal injury is related to elevation of intracellular cyclic-3',5'-adenosine monophosphate.

PG of the E series inhibit major effector functions of polymorphonuclear leukocytes (PMN) by elevating intracellular cAMP. The present study investigated the involvement of this mechanism in the bactericidal defect of PMN induced by thermal injury in a guinea pig model. Peripheral and peritoneal exudate PMN harvested from thermally injured guinea pigs at 1 or 2 days postburn had decreased bactericidal activity against Pseudomonas aeruginosa and a marked increase in cAMP content. Production of PGE1 by these cells in the absence of exogenous PMN activators was also increased. Treatment of PMN in vitro or in vivo with nonsteroidal anti-inflammatory drugs (indomethacin, ibuprofen, and piroxicam) restored bactericidal activity to normal and concomitantly reduced cAMP content and PGE1 production. A concomitant reduction in cAMP content and PGE1 production was also observed as bactericidal activity of PMN returned to normal under natural conditions during 4 to 7 days postburn. The enhancement of PMN bactericidal activity mediated by NSAID was fully counteracted by purified PGE1, theophylline, and by cAMP itself. These results suggest that the bactericidal defect of PMN induced by thermal injury is related to elevation of cAMP and that PGE1 plays a significant role in this phenomenon.

Oral N-acetylcysteine reduces selected humoral markers of inflammatory cell activity in BAL fluid from healthy smokers: correlation to effects on cellular variables.

Bronchoalveolar lavage (BAL) was performed on eleven healthy smokers before and after eight weeks of oral treatment with N-acetylcysteine (NAC) 200 mg t.i.d. The concentrations of selected eosinophil and neutrophil granule constituents and of selected proteases and protease inhibitors, albumin and endotoxin were determined in the recovered BAL fluid and in plasma or serum samples. In addition, in vitro chemotactic activities for neutrophils and eosinophils were assessed in the BAL fluid. Significant reductions in BAL fluid content of lactoferrin (LF), eosinophil cationic protein (ECP), antichymotrypsin (ACT) and chemotactic activity for neutrophils were recorded after NAC treatment. The levels of other examined markers tended to be reduced or were not affected. In serum/plasma, the concentrations of myeloperoxidase (MPO) and elastase were reduced after NAC treatment whereas concentrations of other constituents examined were unaltered. These data, together with previously reported findings, suggest that oral NAC may influence the activity of "inflammatory" cells in the bronchoalveolar space of smokers.

Nonsteroidal anti-inflammatory drugs correct the bactericidal defect of polymorphonuclear leukocytes in a guinea pig model of thermal injury.

We conducted studies to determine the effects of parenteral therapy with indomethacin, ibuprofen, and piroxicam on key immunologic and hematologic alterations induced by thermal injury. Drugs (10-20 mg/kg) or placebo were administered intramuscularly to thermally injured guinea pigs at 3 h postburn and then daily for nine days postburn. All three drugs inhibited production of 6-keto prostaglandin F1 alpha and thromboxane B2 in wound fluid and concomitantly restored the bactericidal activity of polymorphonuclear leukocytes (PMNLs) against Pseudomonas aeruginosa to normal. Indomethacin also increased the proliferative response of splenic lymphocytes to concanavalin A; however, ibuprofen and piroxicam had no effect on this response. None of the drugs affected the extent of systemic complement consumption, thrombocytopenia, leukocytosis, or leukopenia in the injured animals. These results suggest that the PMNL bactericidal defect induced by thermal injury is preventable or reversible and that the mechanisms responsible for this defect are inhibitable by nonsteroidal anti-inflammatory drugs.

Opsonization of bacteroides by the alternative complement pathway reconstructed from isolated plasma proteins.

Opsonization of clinical isolates of B. fragilis and B. thetaiotaomicron with the six isolated proteins of the alternative complement pathway under physiological conditions resulted in considerable C3 deposition on the bacterial surfaces. The time course of C3 deposition was similar to that observed in EGTA-serum; however, the magnitude of C3 deposition was twofold greater in EGTA-serum. Opsonization of the bacteria with the isolated alternative pathway proteins failed to promote adherence, uptake, or killing by polymorphonuclear leukocytes, whereas opsonization of the bacteria with EGTA-serum facilitated these events. The difference in opsonic capacity of isolated proteins and EGTA-serum was not related to the quantitative difference in C3 deposition, because repeated opsonization of the bacteria with isolated proteins resulting in C3 deposition comparable to that observed in EGTA-serum only minimally increased adherence of the bacteria to polymorphonuclear leukocytes. SDS-PAGE and autoradiographic analysis of C3 extracted from bacteria opsonized with isolated proteins or EGTA-serum using methylamine and SDS demonstrated that the predominant form of C3 bound by ester bonds under both sets of conditions was iC3b. A low molecular weight C3 cleavage fragment was detected in extracts from bacteria opsonized with isolated proteins, but it accounted for only a minor fraction of the bound C3. The results of our study demonstrate that the early phase of opsonization involving activation of the alternative pathway by B. fragilis and B. thetaiotaomicron and resultant C3 deposition on the bacterial surfaces does not require auxiliary serum factors, but the effector phase of opsonization of these bacteria involving recognition of bacteria-bound C3 by polymorphonuclear leukocytes and the induction of phagocytosis and intracellular killing is dependent on such factors. Natural IgM antibodies serve as auxiliary factors is opsonization of B. thetaiotaomicron by the alternative pathway, whereas additional serum factors are required for alternative pathway-mediated opsonization of B. fragilis.

Direct evidence that decreased serum opsonization of Streptococcus pneumoniae via the alternative complement pathway in sickle cell disease is related to antibody deficiency.

Two approaches were used to demonstrate that reduction in serum opsonization of Streptococcus pneumoniae via the alternative complement pathway in children with sickle cell disease is related to a deficiency of antibodies to pneumococcal capsular polysaccharide. First, opsonization of S. pneumoniae mediated by the alternative pathway in patients' sera was restored to normal by addition of the purified IgG or IgM fraction of goat antiserum to capsular polysaccharide of the homologous serotype. Secondly, IgG antibody titers to capsular polysaccharide in patients' sera correlated significantly with alternative pathway-mediated opsonization; the correlation between titers of IgM anticapsular antibodies and opsonization approached statistical significance. The sum of the IgG and IgM anticapsular antibody titers correlated most significantly with opsonization. Our results suggest that reduction in alternative pathway-mediated opsonization in sera from children with sickle cell disease is related to low levels of both IgG and IgM anticapsular antibodies.

Lack of a requirement for the Fc region of IgG in restoring pneumococcal opsonization via the alternative complement pathway in sickle cell disease.

Children with sickle cell disease have reduced serum opsonization of Streptococcus pneumoniae. Our previous studies have suggested that opsonization mediated by both the alternative and classic complement pathways is reduced because of a deficiency of IgG antibodies to pneumococcal capsular polysaccharide. This study compares the ability of purified IgG (fractionated from goat antiserum to pneumococcal capsular polysaccharide) and F(ab')2 fragments of the IgG preparation to restore alternative pathway-mediated opsonization of S. pneumoniae to sera from patients with sickle cell disease. Both the whole IgG preparation and F(ab')2 fragments of this preparation restored opsonization to normal levels and concomitantly increased alternative pathway-mediated deposition of C3 onto the pneumococci to a supranormal level. These results suggest that enhancement of opsonization is mediated by the F(ab')2 region of IgG antibody to capsular polysaccharide and is associated with an increase in complement deposition on the bacterial surface.